Hi all, I'm using cufflinks to look at differential isoforms and splicing, but I've come across this problem that cuffdiff doesn't perform the differential splicing tests along with many other tests. The pipeline I use is:
STAR alignment (output XS attribute) -> cufflinks -> stringtie -> cuffquant -> cuffdiff. See some of the details below:
Since I have 41 RNA-seq libraries and I need to merge all "transcripts.gtf" and cuffmerge/cuffcompare is too slow for the task, I took it upon myself to use stringtie to merge all gtf files.
Then I used cuffquant to generate cxb files for each libraries as running cuffdiff directly takes forever, my code looks like this:
cuffquant -p 20 -o 1 stringtie_merged.gtf STAR.cufflinks/sample_1/Aligned.sortedByCoord.out.bam
Afterwards, I used cuffdiff to perform differential testing:
cuffdiff -p 20 -L cond1,cond2,control -o cuffdiff_pc stringtie_merged.gtf 1/abundances.cxb,2/abundances.cxb,3/abundances.cxb 4/abundances.cxb,5/abundances.cxb,6/abundances.cxb 7/abundances.cxb,8/abundances.cxb,9/abundances.cxb**
This is what I get:
[03:03:33] Calculating preliminary abundance estimates [03:03:33] Testing for differential expression and regulation in locus. Processed 17291 loci. [*************************] 100% Performed 346094 isoform-level transcription difference tests Performed 0 tss-level transcription difference tests Performed 96730 gene-level transcription difference tests Performed 0 CDS-level transcription difference tests Performed 0 splicing tests Performed 0 promoter preference tests Performing 0 relative CDS output tests Writing isoform-level FPKM tracking Writing TSS group-level FPKM tracking Writing gene-level FPKM tracking Writing CDS-level FPKM tracking Writing isoform-level count tracking Writing TSS group-level count tracking Writing gene-level count tracking Writing CDS-level count tracking Writing isoform-level read group tracking Writing TSS group-level read group tracking Writing gene-level read group tracking Writing CDS-level read group tracking Writing read group info Writing run info /var/spool/torque/mom_priv/jobs/5102221.torque01.util.production.int.aci.ics.psu.edu.SC: line 15: 160872 Segmentation fault (core dumped) cuffdiff -p 20 -L cond1,cond2,control -o cuffdiff_pc stringtie_merged.gtf 1/abundances.cxb,2/abundances.cxb,3/abundances.cxb 4/abundances.cxb,5/abundances.cxb,6/abundances.cxb 7/abundances.cxb,8/abundances.cxb,9/abundances.cxb
Notice that there's a core dumped error but I don't think this has anything to do with memory issue as I'm running the commands on a HPC.
Any ideas would be appreciated,